Effluent collection apparatus and method

ABSTRACT

The present invention includes a method of analyzing one or more molecular components in a mixture of components. In the method, a mixture of molecular components, i.e., an analyte mixture, is separated on a capillary liquid chromatography column. The component-containing eluate from the column is deposited as a series of discrete, defined-volume microdrops, along a region of an adsorbent collection layer. During the chromatographic separation, the column eluate may also-be monitored to detect the presence of separated components in the equate. The one or more components deposited in the collection layer are then analyzed by selected analytical techniques. In related aspect, the invention includes a method of collecting one or more molecular components derived from a mixture of components, a blotter apparatus useful in the above method, and a system for analyzing one or more molecular components in a mixture of components.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/885,292, filed Jun. 19, 2001, now U.S. Pat. No. 6,582,547, which is acontinuation of U.S. application Ser. No. 08/887,350, filed Jul. 2,1997, now U.S. Pat. No. 6,248,239 which claims priority under 35 U.S.C.120 to provisional patent application No. 60/016,095, filed Jul. 8,1996, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods of fractionating and collectinganalytes. In one embodiment, the invention relates to an improved methodfor collecting chromatographically separated analytes such aspolypeptides, polynucleotides, and polysaccharides.

REFERENCES

Fernandez, J., et al., Anal. Biochem. 201:255 (1992).

Hawke, D. H. and Yuan, P-M, Applied Biosystems User Bulletin 28:1-8(1978, 1988).

Kochersperger, M. L., et al., Protein Science 3:Suppl. 1 98, 265-M(1994).

Kuo-Liang Hsi, et al., in Techniques in Protein Chemistry IV pp. 143-150(1993).

Sambrook, J., et al., Molecular Cloning, 2nd Ed., Cold Spring HarborLaboratory Press (1989).

Murata, H., et al., Anal. Biochem. 210:206 (1993).

Spellman, W. M., et al., J. Biol. Chem. 264:14100 (1989).

BACKGROUND

Purification and characterization of chemical analytes such aspolypeptides, polynucleotides, and polysaccharides, have becomeincreasingly important in the chemical and medical arts. Numerousanalytical methods have been developed for a variety of purposes, suchas testing for the presence of biological contaminants or toxins,identifying new components in biological systems, and verifying samplepurity, for example. Often, analytes of interest are available only intrace amounts or at very low concentrations. Accordingly, there has beenmuch interest in developing analytical techniques with increasedsensitivity to facilitate characterization of such analytes.

For many applications, one or more purification steps are necessarybefore the analyte(s) of interest can be detected or quantified. In thecase of analytes which are present in trace amounts, purification hasproved difficult for a number of reasons. For example, when analyteselute closely together under given separation conditions, it has beendifficult to collect adjacent peaks in a manner that retains resolution,i.e., without significantly diminishing the resolution achieved by theselected purification method. Such small-sample purifications have alsobeen hampered by low sample recoveries due to dilution or adherence ofsample on collection vessel surfaces.

Although fraction collection using individual collection vessels hasbeen the traditional mode for collecting and storing resolved samplecomponents, this approach has generally been unsuitable for small sampleamounts because of low recoveries as above. Accordingly, othercollection methods have been proposed.

One proposed approach involves collecting eluted samples on an adsorbentsurface by continuously dragging the outlet of a chromatography columnacross an adsorbent surface, such that the column effluent iscontinuously dispensed onto the adsorbent in a continuous trail. Such amethod has been proposed by Murata et al. (1993) for collectingpolypeptides from a capillary liquid chromatography column. In theirmethod, a pen-holding device is used to maintain the column outlet incontinuous contact with a collection membrane.

Although such dragging methods have allowed relatively simple apparatusdesign, subsequent experience has shown that the fluid outlet oftensnags on the adsorbent surface, leading to tearing or gouging of thesurface or, conversely, locking of the outlet onto the surface so thatthe outlet cannot move or the adsorbent moves with the outlet. Temporarycatching of the outlet on the membrane can lead to discontinuities andother irregularities in the deposited sample, so that the locations ofresolved peaks do not correspond with the true elution profile. Tearingor gouging can seriously hinder sample recovery. Long-term lockingbetween the surface and the outlet can result in superimposition of someor all of the resolved peaks, defeating the purpose of the separation.Yet another drawback of the dragging method is the possibility ofcross-contamination of eluate due to carry-over of liquid between thecapillary outlet and the adsorbent collection layer.

It would be desirable to provide an apparatus and method for collectingsmall amounts of eluted sample components with high sample recovery,while avoiding the problems mentioned above. In particular, it would bedesirable to provide such a method for collecting separated samplecomponents in a manner that allows immediate use or long-term storagefor subsequent analysis.

SUMMARY OF THE INVENTION

The present invention is directed, in one aspect, to a method ofanalyzing one or more molecular components in a mixture of components.In the method, a mixture of molecular components, i.e., an analytemixture, is separated on a capillary liquid chromatography column. Thecomponent-containing eluate from the column is deposited as a series ofdiscrete, defined-volume microdrops, along a region of an adsorbentcollection layer. During the chromatographic separation, the columneluate may also be monitored to detect the presence of separatedcomponents in the eluate. The one or more components deposited in thecollection layer are then analyzed by selected analytical techniques.

In one embodiment of the method, the collection layer is immobile duringthe depositing step, and the depositing step includes reciprocating adeposition head, for depositing the eluate on the collection layer,toward and away from a position of contact with the collection layer,while the deposition head is moved laterally relative to the collectionlayer. In a preferred embodiment, the deposition head is moved laterallyover the collection layer in a linear direction. In an alternativeembodiment, the depositing step includes reciprocating a deposition headtoward and away from a position of contact with the collection layerwhile the collection layer is moved laterally relative to the depositionhead.

In related aspect, the invention includes a method of collecting one ormore molecular components derived from a mixture of components. In themethod, a mixture of molecular components is separated on a capillaryliquid chromatography column. The component-containing eluate from thecolumn is deposited as a series of discrete, defined-volume microdropsalong a region of an adsorbent collection layer. The collectedcomponents in the collection layer may then be analyzed by selectedanalytical techniques.

In another aspect, the invention includes a blotter apparatus which isuseful in the methods described above. The apparatus includes anadsorbent collection layer, and means for depositingcomponent-containing eluate from a capillary liquid chromatographycolumn as a series of discrete, defined-volume microdrops, along aregion of the adsorbent collection layer. In one embodiment, theapparatus further includes means for monitoring the column eluate todetect the presence of separated components in the eluate, and a controlunit operatively connecting the monitoring and depositing means forcontrolling the flow rate and volume of deposited microdrops. Asdescribed above, the collection layer may be immobile, and thedepositing means includes a deposition head which is capable ofreciprocating toward and away from a position of contact with thecollection layer while the deposition head is moved laterally relativeto the collection layer. In an alternative embodiment, the deposition isimmobile with respect to lateral movement, and the apparatus includesmeans for moving the deposition layer laterally relative to thedeposition head.

The invention also includes a system for analyzing one or more molecularcomponents in a mixture of components. The system includes (i) acapillary liquid chromatography column, (ii) means supplying liquid tothe column at a selected flow rate, (iii) means for monitoring thecolumn eluate to detect the presence of separated components in theeluate, (iv) means for depositing component-containing eluate from thecolumn as a series of discrete, defined-volume microdrops, along aregion of an adsorbent collection layer, and (v) a control unitoperatively connecting the monitoring and depositing means forcontrolling the flow rate and volume of deposited microdrops.

In one embodiment, the depositing means includes (i) a stage adapted tosupport the adsorbent collection layer, (ii) a deposition head operableto reciprocate toward and away from a position of contact with thecollection layer, and (iii) means for moving the stage and headlaterally with respect to one another.

In one embodiment, the stage is effective to hold the collection layerimmobile, and the depositing means includes means for moving thedeposition head laterally with respect to the collection layer. In analternative embodiment, the deposition head is immobile with respect tolateral movement, and the system further including means for moving thestage laterally with respect to the deposition head.

The control means may be operable to change the deposition rate andmicrodrop deposition volume in response to different peak patternsdetected by the monitoring means.

These and other objects and features of the invention will be moreapparent from the following detailed description when read in light ofthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a simplified schematic view of a blotting apparatus whichmay be used in practicing the invention;

FIG. 2 shows an exploded view of a dynamic blotter element for raisingand lowering the outlet end of a capillary tube above the surface of anadsorbent collection layer;

FIG. 3 shows an overhead view of a tray assembly for use in the blotterapparatus of the invention;

FIG. 4 shows schematically a blotting system in accordance with thepresent invention;

FIG. 5 shows a chromatogram of a sample mixture containing ribonucleaseA (peak 1), lysozyme (peak 2), and apomyoglobin (peak 3);

FIG. 6 shows a chromatogram of a tryptic digest of bovine serum albumin;

FIG. 7A shows a chromatogram of a tryptic digest of t-PA;

FIG. 7B shows a chromatogram of glycopeptides isolated by lectinaffinity binding; and

FIG. 8 shows a chromatogram of a tryptic digest of apomyoglobin.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The following terms as used herein are intended to have the followingmeanings unless the context indicates otherwise.

“Capillary liquid chromatography” or “cLC” refers to low and highpressure chromatographic methods in which the chromatography column andassociated tubing for transporting solvents and eluate are of smallinternal cross sections, typically of 1 mm or less, more typically lessthan 200 μm, and preferably less than 100 μm.

II. Apparatus

In one aspect, the invention includes a blotter apparatus useful forcollecting one or more chromatographically separated molecularcomponents from a mixture of components. The apparatus includes anadsorbent collection layer, and means for depositingcomponent-containing eluate from a capillary liquid chromatographycolumn as a series of discrete, defined-volume microdrops, along aregion of the adsorbent collection layer.

FIG. 1 illustrates an exemplary blotter apparatus 20 for use in theinvention. Apparatus 20 includes means 22 for depositing eluate from acapillary liquid chromatography (cLC) column onto an adsorbentcollection layer 34. As illustrated in the figure, depositing means 22is a deposition head which includes a “push-pull” solenoid element 23,and passing through the head, outlet region 24 of the cLC column. The“push-pull solenoid” is also referred to herein as “means capable ofreciprocating toward and away from a position of contact with thecollection layer”. The deposition head is carried by arm 26 whichprotrudes out of slot 30, for laterally moving the deposition head overthe collection layer. Lateral movement of arm 26 may be achieved using astepper motor assembly (e.g., screw-type) according to mechanisms knownin the art.

Electrical signals from a controller (not shown) for reciprocating thehead towards and away from the adsorbent layer are transmitted to thesolenoid element via electrical connection 28. Through the reciprocatingaction of the head, cLC outlet end 32 is brought into contact withcollection layer 34 to deposit the eluate as a series of discrete,defined-volume microdrops along a region of collection layer 34.

The apparatus further includes a sample tray 36, which may be referredto as a “stage adapted to support the adsorbent collection layer”, andwhich fits snugly in a complementary cavity which holds the tray andadsorbent immobile during operation. The sample tray, adsorbent layer,and deposition head are also encompassed by the term “means for movingthe stage and head laterally with respect to one another.”

With continued reference to FIG. 1, the apparatus further includes acontrol panel 38 for setting the lateral speed of the deposition headand optionally, the rate of reciprocation of the head. The apparatus mayalso include a recess region 40 for storing the head in a non-exposed“home position” when eluate is not being collected.

A depositing means 22 in accordance with FIG. 1 is illustrated infurther detail in FIG. 2. Depositing means 22 includes a solenoid 50equipped with an electrical connection 52 for delivering current to thesolenoid. Solenoid 50 further defines a hollow cylindrical region alongand coaxial with its longitudinal axis for receiving core 54.

Core 54 includes a wire spring 56, washer 58, and C-ring 60, which arefitted around the core to provide a spring mechanism as describedfurther below. The core 54 and its elements 56, 58 and 60 are enclosedby means of a bushing 62 a having an internal female screw-type fitting(not shown) for snugly engaging a corresponding male fitting 62 b on thelower portion of solenoid 50.

Capillary outlet region 64 (corresponding to outlet region 24 in FIG. 1)passes through the longitudinal axes of solenoid 50, core 54, andbushing 62 a so that capillary outlet end 66 extends through the bottomof bushing 62 a. Outlet region 64 is snugly held by the interior of core54, but not by solenoid 50 or bushing 62 a. This allows the verticalmotion of capillary outlet end region 66 to be controlled by thevertical movement of the core within the confines of the solenoid andbushing. It will be appreciated that although capillary outlet region 64is shown as passing through the center of the deposition head, theoutlet may be located elsewhere on the deposition head, e.g., on theexterior of the solenoid element with appropriate linkage to core 54 toachieve controlled vertical movement of the outlet.

Outlet end 68 is capped snugly with a sleeve 68, preferably made of“TEFLON”, which is made flush with the tip of outlet end 66. Sleeve 68helps maintain the eluate in the region immediately below the tip bypreventing the eluate from climbing up the outside of the outlet end anddisrupting fluid flow. The sleeve is also effective to cover the sharpedges of the capillary tip so as to shield the adsorbent layer frompotential snagging or tearing. A further advantage of the sleeve is thatthe sleeve increases the effective cross-section of the capillary outletso that the impact of the outlet on the adsorbent layer is dampened,preventing puncture of the adsorbent layer.

When fully assembled, deposition head 22 can reciprocate between twopositions designated here as the extended position (contact with theadsorbent layer) and the retracted position (no contact with theadsorbent layer). In the resting state, i.e., when no current issupplied to the solenoid, deposition head 22 occupies the extendedposition due to the pushing force of spring 56 acting against the tip ofmale fitting 62 b. When the head is positioned over the adsorbentcollection layer, outlet end 66 and sleeve 68 are brought into contactwith the adsorbent layer. Upon contact, any eluate that has collectedbeneath the tip of outlet end 66 is deposited onto, and adsorbed by, thecollection layer.

It will be appreciated that the force with which the tip of the outletcontacts the adsorbent collection layer can be adjusted and minimized bysuitable choice of spring stiffness, and by positioning the depositionhead relative to the surface of the adsorbent collection layer so thatthe capillary outlet tip only gently touches the adsorbent layer whenthe head is in the extended (contact) position. As noted above, theimpact of the outlet tip on the collection layer is further softened bythe presence of sleeve 68. During contact with the adsorbent layer, theeluate is drawn from the tip into the adsorbent layer.

The deposition head is moved to the retracted position by passage ofcurrent through solenoid 50 via connection 52. The magnetic fieldgenerated in the interior of the solenoid draws core 54 upward towardthe solenoid, so that outlet 66 is lifted away from the collectionlayer. During the time the head is in the retracted position, eluatecollects under the tip of capillary outlet region 66 until the next timethe tip is contacted with the adsorbent layer.

FIG. 3 illustrates a linear adsorbent layer and tray assembly (i.e., astage adapted to support the adsorbent collection layer) which may beused in the apparatus of FIG. 1. Assembly 70 includes an adsorbent layer34 which is held in a shallow depression (not shown) provided by tray36, which immobilizes the layer from lateral movement. The assemblyfurther includes a positioning notch 72 which interlocks with acorresponding protrusion in the blotter apparatus to ensure properorientation. The tray may also include a mark 74 which indicates thestart site for sample collection from the deposition head.Alternatively, the adsorbent layer may include such a mark if desired.For the device described in the Example section below, the overheaddimensions of the adsorbent layer were approximately 1.8 mm×200 mm. Ofcourse, these dimensions are merely illustrative.

The adsorbent collection layer is formed of any adsorbent materialappropriate for the analyte of interest. Where the analyte is a protein,the adsorbent is preferably one which binds protein with a strongaffinity. Exemplary adsorbent materials include polyvinylidenedifluoride (PVDF), nitrocellulose, and various other membranes in theImmobilon series available from Millipore (Bedford, Mass.). For bindingof polynucleotides, nitrocellulose and nylon are useful, for example(e.g., available from Schleicher & Schuell, Keene, N.H.). The collectionlayer may also have additional layer underneath the layer which contactsthe capillary outlet, to help draw the eluate solvent into thecollection layer.

The rate of lateral movement of the deposition head with respect to theadsorbent layer, the frequency of contact, and the durations of contactand non-contact between the capillary outlet and adsorbent layer willdepend on various factors, such as the flow rate of eluate, the degreeof resolution of sample components to be collected, the amount of thecomponents, and the absorptive capacity of the adsorbent layer. Forcapillary liquid chromatography, flow rates are generally between 1 and100 μL/min, and preferably between 1 and 10 μL/min. In the examplesdescribed below, the contacting cycle time was 2 seconds per cycle, witha contact time of 1.8 seconds and a non-contact time of 0.2 seconds. Thelateral speed of the deposition head relative to the adsorbent layer was1 mm/min. At a flow rate of 4 μL/min, with 30 contact cycles per minute,the volume of eluate deposited per contact cycle was about 130 nL, witha density of 4 μL deposited per mm of adsorbent. It will be appreciatedthat other deposition rates and lateral speeds may be used, according tothe requirements of the particular sample and separation conditions.

While the arrangement of the adsorbent layer and deposition pen areshown with regard to a particular embodiment in FIGS. 1 to 3 (lateralmovement of a reciprocating deposition head in a linear direction overan immobile adsorbent layer), it will be appreciated that otherconfigurations can also be used. For example, a reciprocating depositionhead can be positioned at the end of an arm which rotates about an axisperpendicular to the plane of the adsorbent layer. In thisconfiguration, the eluate is collected in a circular or arc shapedpattern. When eluate can also be collected in a spiral pattern on theadsorbent layer when the apparatus includes means for shortening thelength of the holding arm.

In an alternative embodiment, the reciprocating deposition may be heldimmobile with respect to lateral movement, and the apparatus includesmeans for moving the adsorbent layer laterally relative to thedeposition head. In one embodiment, the adsorbent layer can be movedlaterally with respect to the deposition head by means of a take-upwheel which pulls the adsorbent layer past the head, where axis ofrotation of the wheel is perpendicular to the longitudinal axis of thedeposition head and parallel to the surface of the adsorbent layer.Alternatively, the apparatus includes means for rotating the adsorbentlayer about an axis parallel to the longitudinal axis of the depositionhead, so that the eluate is collected in a circular or arc-shapedpattern. Other configurations are also possible.

In an alternative embodiment, the depositing means of the apparatus isimmobile with respect to vertical movement (movement in a directiontoward and away from the adsorbent layer), and contact between thecapillary outlet and the adsorbent layer is accomplished byreciprocating movement of the adsorbent layer toward and away from aposition of contact with the capillary outlet of a nonreciprocatinghead. In one embodiment, the apparatus includes means for moving theadsorbent layer in a reciprocating fashion toward and away from thedeposition head, as just mentioned, and also means for moving theadsorbent layer laterally with respect to the adsorbent layer.Alternatively, the apparatus includes means for moving the adsorbentlayer in a reciprocating fashion with respect to a nonreciprocatingdeposition head, and means for moving the deposition head laterally withrespect to the adsorbent layer along the lines discussed above.

In a further embodiment, the apparatus includes means for monitoring thecolumn eluate to detect the presence of separated components in theeluate, and a control unit operatively connecting the monitoring anddepositing means for controlling the flow rate and volume of depositedmicrodrops. The monitoring means include a detector which is selectedaccording to the signal of the sample components to be detected, asdiscussed further below. Typical detection methods include UV-visibleabsorption, fluorescence, chemiluminescence, CCD, radioisotopicdetection, and ionization detection (electrical conductance), forexample.

III. Collection System

The invention also includes a system for analyzing one or more molecularcomponents in a mixture of components. The system includes (i) acapillary liquid chromatography (cLC) column, (ii) means supplyingliquid to the column at a selected flow rate, (iii) means for monitoringthe column eluate to detect the presence of separated components in theeluate, (iv) means for depositing component-containing eluate from thecolumn as a series of discrete, defined-volume microdrops, along aregion of an adsorbent collection layer, and (v) a control unitoperatively connecting the monitoring and depositing means forcontrolling the flow rate and volume of deposited microdrops.

FIG. 4 illustrates an exemplary system 80 in accordance with theinvention. System 80 includes solvent pump means 82 which draws solventfrom reservoirs 84 a and 84 b at selected flow rates and ratios, andwhich are mixed together by mixer 86. The system also includes a sampleinjector 88 equipped with a sample loop, vent, and waste lines asconventional for cLC. Pump means 82 and reservoirs 84 a,84 b arecollectively referred to as “means for supplying liquid to the cLCcolumn at a selected flow rate”. The sample injector is connected bysuitable tubing connections to cLC column 90. Eluate from column 90passes through detector 92, whose signal output is displayed on a chartrecorder 94 (or equivalent display device). Eluate exiting from thedetector is fed to a depositing means or blotter apparatus 20.

The depositing means 20 preferably includes (i) a stage adapted tosupport the adsorbent collection layer, (ii) a deposition head operableto reciprocate toward and away from a position of contact with thecollection layer, and (iii) means for moving the stage and headlaterally with respect to one another. These features are substantiallyas described above with respect to the various depositing meansdescribed in section II.

The cLC column, pump means, solvents, and solvent gradient are selectedaccording to the analyte components to be collected.

The cLC column used in the system may be any cLC column available in theart. A large variety of cLC columns are available from numerouscommercial suppliers, including J&W Scientific, Micro-Tech Scientific,Interchim, Alltech, Supelco, Hewlett-Packard, and others. The column maycontain any suitable solid phase, such as normal phase (e.g., silicagel), reversed phase (e.g., C-18 or C-8 derivatized supports),hydrophobic interaction, cation and anion exchange resins, immobilizedantibody affinity columns, and the like. The columns are usually smallerthan standard LC columns, typically having inner diameters of 0.2 to 1.0mm and lengths of 5 to 25 cm, although columns having dimensions beyondthese ranges are also contemplated.

For charged analytes such as polynucleotides and proteins, anion orcation-exchange solid supports may be used, where elution is via saltgradient or change in pH. Alternatively, reversed-phase supports arealso effective, where elution is accomplished using a gradient ofincreasing or decreasing organic solvent concentration, usually in thepresence of an ion-pairing reagent such as TFA (trifluroacetic acid) orvarious ternary or quaternary amine compounds, as are known in the art.For separation of organic compounds, such as aromatic compounds, organictherapeutics, hydrocarbons, esters, ethers, amine-containing compounds,carboxylic acids, organic polymers, and the like, normal phase, ionexchange, or reversed phase supports may be appropriate.

The solvent pump is any pump capable of delivering solvent at a flowrate suitable for the selected purification method, including anisocratic or gradient format as desired. For typical peptideseparations, flow rates of about 1 to 100 μL/min are generally suitable,with flow rates of about 1 to 10 μL/min being preferred for manymicrocolumn applications. Flow rates outside these ranges may also beappropriate, depending on the desired peak resolution and/or otherfactors.

Solvent pumps are selected to provide suitable flow rates and gradientproperties. Such pumps are known in the art and may be obtained fromvarious commercial suppliers. A preferred pumping system is the 140Ddual syringe pump available from Perkin-Elmer, which consists of two 2.5mL syringe pumps under the control of a gradient controller. This pumpsystem provides pulse-less flow rates as low as about 1 μL/min atpressures as high as 3000 psi. Further description of a pump useful forthe present invention can be found in copending U.S. application Ser.No. 08/414,663 filed Mar. 31, 1995, the disclosure of which isincorporated herein by reference.

Tubing connections are preferably configured to minimize dead volume inthe system and to be inert with respect to the solvents and analytesused in the separation. Fused silica transfer lines, “TEFLON” tubing,and stainless steel are suitable for most applications, although othermaterials may be used.

The sample injector is likewise conventional. For example, a 112A SampleInjector from Perkin-Elmer may be used. The sample loop for loading thesample onto the column typically holds a sample volume of 1 to 50 μL,and preferably 5 to 25 μL, according to the amount and concentration ofthe analytes to be measured.

The eluate monitoring means is any detector appropriate for monitoringthe analytes of interest. The detector operates according to anydetection technique that is appropriate for the analytes of interest.Typical detection methods include UV-visible absorption, fluorescence,chemiluminescence, CCD, radioisotopic detection, and ionizationdetection (electrical conductance), for example.

The analytes of interest are detected on the basis of an intrinsicallydetectable signal, or may be derivatized with a label which confers adesired type of detectability. Various methods for labeling analyteswith detectable moieties are well known in the art, such as radioactiveisotopes, fluorescent dyes, spin labels, chemiluminescent compounds, andthe like. When the analyte is a polynucleotide, labeling byhybridization with a labeled probe is also contemplated.

Detection by UV-visible absorbance spectroscopy is accomplished byconventional techniques. The detector includes a light source which maybe of variable or constant wavelength and bandwidth. The light beam isfocussed by appropriate means, e.g., a spherical sapphire lens, tominimize dispersion. Preferably, the detector employs a standarddual-beam arrangement which allows cancellation of output fluctuationsof the light source. An exemplary absorbance detector which may be usedin the system is a 785 A Programmable Absorbance Detector available fromPerkin Elmer, Applied Biosystems Division (Foster City, Calif.).

The detector further includes a flow cell configured to providesufficient sensitivity for the signal to be detected or measured.Conveniently, the flow cell is a U-shaped flow cell of selected pathlength and cross-section, which allows an illuminating light source topass down the path of fluid flow through the bottom arm of the U-cell.Typical path lengths are from about 2 to 10 mm. Typical cross-sections(inner diameters) are generally from about 25 to about 200 μm. It willbe appreciated that the parameters of the flow cell will depend on theproperties of the analytes to be detected.

The eluate depositing means is substantially as described with referenceto the blotter apparatus described in Section II.

The control unit of the system operatively connects the monitoring anddepositing means for controlling the flow rate and volume of depositedmicrodrops. The control unit may be prepared using standardmicroprocessor components and design. The control unit accepts datainput from the user regarding the start of sample collection and thestart of signal recordation, as well as chart recorder speed and lateralspeed of the deposition head relative to the adsorbent layer. Thecontroller also regulates the reciprocating rate of the head relative tothe adsorbent layer and the duration of contact between the adsorbentlayer and the capillary outlet in each contact cycle.

The control means may also be operable to change the deposition rate andmicrodrop deposition volume in response to different peak patternsdetected by the monitoring means. For example, the control means can beprogrammed to select, for analysis of selected components, those dropsand only those drops that contain those components in pure form, thusmaximizing the amount of pure material available for analysis andconserving the collection layer. The control means can also reducedroplet size and increase deposition rate to improve separation of anycomponent during a run. Finally, the control means can be programmed tocoordinate eluate monitoring and microdrop deposition to minimizepeak-component overlap in the deposited microdrops.

IV. Method

The present invention includes a method of collecting one or moremolecular components derived from a mixture of components. In themethod, a mixture of molecular components is separated on a capillaryliquid chromatography column. The component-containing eluate from thecolumn is deposited as a series of discrete, defined-volume microdropsalong a region of an adsorbent collection layer, using an apparatus orsystem as described above. The collected components in the collectionlayer may then be analyzed by selected analytical techniques. In thisregard, the invention also includes a method of analyzing one or moremolecular components in a mixture of components, as outlined above.

In the method of the present invention, the collected sample componentsmay be analyzed by a variety of methods. For protein samples, individualprotein components (e.g., intact proteins or proteolysed peptidefragments) can be excised from the collection layer using a razor blade,for transfer to a protein sequencer. In this regard, it is particularlyuseful to have obtained a chromatogram of the eluate to allow alignmentof the eluate with the collection layer to identify the positions of theseparated components on the collection layer, as illustrated in theExamples below. Such protein samples may also be analyzed while stillbound to the collection layer, e.g., by immunoassay to detect thepresence or amount of selected analytes, or in a binding assay with alabeled receptor or ligand which is binds the analyte.

Collected sample components may also be transferred by blotting oroverlay with solid gel layers and the like (e.g., polyacrylamide gels)which contain enzymes substrates, immunoassay components, or othersuitable reagents for producing a signal in the presence of the selectedanalytes. For examples, Northern, Southern, and western blottingtechniques may be used as appropriate, according to methods known in theart (e.g., Sambrook, 1989).

The features of the invention will be further appreciated fromdiscussion of Examples 1 to 4 below, with reference to FIGS. 5 to 8.

FIG. 5 illustrates use of the invention for the collection and analysisof polypeptides. As detailed in Example 1, a protein mixture containing0.3 μg each of ribonuclease A, lysozyme, and apomyoglobin in 0.1% TFAwas chromatographed using a C-18 microcolumn with an acetonitrilegradient, and the effluent was collected on a PVDF membrane. Whenchromatography was complete, the PVDF membrane was dried, washed withmethanol and water, and soaked for 1 to 2 minutes in a protein stainingsolution consisting of 0.05% (w:v) copperphthalocyanine-3,4′4″4′″-tetrasulfonic acid tetrasodium salt (CPTS) in0.1% TFA. After staining, the membrane was destained with water andallowed to dry. The three stained spots, which corresponded to theexpected protein peaks on the chromatogram, were excised from themembrane and transferred to a sequencer cartridge for sequencing. Asseen from Table 1 in Example 1, initial sequencing yields were high,ranging from 56% to 62%.

Examples 2 to 4 illustrate the usefulness of the invention for analysisof peptide fragments from a range of proteins. In Example 2, a trypticdigest of BSA (10 pmol) was chromatographed on a C-18 column with ashallower acetonitrile gradient. As a visible standard, the sampleincluded methyl violet base B (“dye marks”) which produced a triplet ofpeaks that eluted after the last of the tryptic fragments (seechromatogram in FIG. 6). Regions of the PVDF membrane corresponding tosix selected peaks (numbered 1 to 6 in FIG. 6) were excised andsubjected to N-terminal sequence analysis. As tabulated in Example 2,initial sequencing yields ranged from about 21% to about 50%, dependingroughly on the length of the peptide sequenced (wash-out effect).

Example 3 describes purification and collection of glycopeptides fromthe glycoprotein t-PA (tissue plasminogen activator). Following trypticdigestion of t-PA, glycopeptides were isolated by lectin affinitybinding (Hsi et al., 1993). Chromatograms of the tryptic digest and thethree affinity-purified glycopeptides are shown in FIGS. 7A and 7B.Initial sequencing yields were about 30-40% for 100 pmol loaded sample,indicating that the cLC-blotter has excellent sample capacity. Thesequencing results showed that the sequences are identical to publishedsequence data for t-PA (Spellman, 1989). Further, each glycopeptidecontained a consensus sequence pattern for N-linked glycosylation,—N—X—S/T, where X is any amino acid and N is glycosylated, whichexplained the blank cycles observed for residues in the N position ineach of the sequenced peptides.

Isolation and analysis of tryptic peptides from apomyoglobin isdescribed in Example 4, with a chromatogram of the fragments shown inFIG. 8. Two peaks, peak 4 (residues 134-139) and peak 6 (residues 64-77)were sequenced, giving high initial yields of 42% and 63%, respectively.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES

Materials and Methods

Chemicals. Bovine serum albumin (BSA), apomyoglobin, reduced “TRITONX-100”, CAPS and “CON A-SEPHAROSE 4B” were purchased from Sigma ChemicalCo. (St. Louis, Mo.). Trypsin was purchased from Promega (Madison,Wis.). Human tissue plasminogen activator (t-PA) was purchased from CalBiochem (San Diego, Calif.). Dye marker methyl violet base B waspurchased from Aldrich (Milwaukee, Wis.). Pre-cast Tris-Tricine gelswere purchased from Novex (San Diego, Calif.). HPLC solvents, TFA(trifluroacetic acid) and “POLYBRENE PLUS” were from Perkin-Elmer,Applied Biosystems Division (Foster City, Calif.).

Peptide Mapping. BSA or t-PA in 0.1 M ammonium bicarbonate was digestedwith trypsin at an enzyme/substrate mass ratio of 1:50 for 10 to 16 hr.Both proteins were reduced and alkylated withβ-mercaptomethanol/4-vinylpyridine as described by Hawke and Yuan (1988)prior to digestion. Digestion of apomyoglobin was carried out in 100 mMTris/HCl buffer containing 10% acetonitrile and 1% reduced Triton X-100.

Preparation of t-PA Glycopeptides. Glycopeptides from t-PA were preparedusing a micro batch lectin affinity binding technique described by Hsiet al. (1993). In brief, a tryptic digest of t-PA was mixed with Con ASepharose 4B in a 1 ml Eppendorf tube for 30 min. After washing withTris-HCl buffer to remove all unbound peptides, the bound glycopeptideswere released with α-methyl mannoside. The resulting glycopeptides,after separation and blotting on the cLC-microblotter, were sequenced ona Procise-HT Sequencer.

Capillary Liquid Chromatoaraphy System. A sample purification andcollection system was assembled as illustrated in FIG. 4. The systemincluded a 140D dual syringe pump (2.5 mL syringe volumes) equipped witha T-mixer; a 112A Injector (Rheodyne 8125) equipped with a 5-20 μLsample loop; a C-18 reversed phase column (150 mm×0.5 mm I.D., 5 μmparticles); a 785A Programmable Absorbance Detector equipped with a 30nL, 6 mm path-length U-shaped flow cell, with signal output directed toa strip chart recorder; and a blotter apparatus equipped with apush-pull solenoid substantially as illustrated in FIGS. 1 and 2, thesolenoid being held by a drive arm for lateral movement over the blottertray. All connective tubing (fused silica) between the pump and thedetector flow cell had an internal diameter (I.D.) of 50 μm and an outerdiameter (O.D.) of about 370 μm. Movement of the solenoid was controlledby a controller which coordinated the lateral speed of the solenoid andthe frequency and duration of contact of the outlet end of the capillarytube with the PVDF membrane.

The capillary tubing connecting the flow cell to the system outlet(i.e., the end protruding from the blotter solenoid) had an I.D. of 30μm. The tip of the capillary tubing protruding from the solenoid wassleeved with a ¼ inch length of 0.012 inch×0.009 inch “TEFLON” tubingwhich was made flush with the protruding end of the capillary tubing.

Prior to each run, the adsorbent tray in the blotter apparatus wasloaded with a strip of filter paper (Biorad Cat. No. 170-3956). Thefilter paper was thoroughly wetted with deionized water. After the traywas tilted to remove excess water from the filter paper, a PVDF membraneof the same size as the filter paper was wetted with methanol and thenplaced directly over the wet filter paper. A dry strip of filter paperwas then laid on top of the PVDF membrane, and a gloved finger was runalong the top of the filter paper from one end to the other to removeair pockets between the PVDF membrane and the underlying filter paper.The top strip of filter paper was then removed, and the blotter tray wasplaced in the blotter housing of the apparatus.

After sample loading and initiation of the solvent gradient program bythe controller, the chart recorder and solenoid were activated so thatthe chart paper and solenoid drive arm moved at the same speeds. Theoutlet end protruding from the solenoid was periodically contacted withthe PVDF membrane as the solenoid moved laterally across the membrane.

When the chromatographic separation was complete, the PVDF membrane wasremoved from the blotter tray with a tweezers and was allowed to air-dryon a “KIMWIPE” tissue. The dried membrane was then aligned with thechromatogram on the chart paper, and membrane regions corresponding topeaks of interest were excised using a razor blade and were usedimmediately for protein sequencing or were sealed in an “Eppendorf” tubeand refrigerated for later analysis.

For the examples below, the blotter speed and strip chart recorder speedwere each 1 mm/min; contact time of the tip of the capillary outlet withthe PVDF membrane was about 1.8 sec per cycle, with a total cycle timeof 2 sec per cycle; solvent A=0.1% TFA in water, solvent B=0.085% TFA inacetonitrile; and absorbance wavelength was 210 nm at 1 AUFS (Example 1)or 0.1 AUFS (Example 2). Collected sample components were typically 1 to2 mm in diameter based on protein staining.

Sequence Analysis. N-terminal sequence analysis was conducted using anApplied Biosystems 473 or Procise-HT Sequencer. All peptides weretreated with 1-2 μL of “POLYBRENE PLUS” solution prior to loading in thesequencer. The “POLYBRENE” solution was made by mixing 1 part of“POLYBRENE PLUS” (100 mg/ml in water), 1 part of 0.1% TFA and 2 parts ofMeOH. Total “POLYBRENE” applied was 25-50 mg per excised membrane piece.

Example 1 Purification and Sequencing of Three Proteins

The system described above was used to purify and collect a sampleconsisting of 0.3 μg each of ribonuclease A, lysozyme, and apomyoglobinin 0.1% TFA. The elution gradient was 15%-65% B over 75 min at a flowrate of 4 μL/min. Once chromatography was complete, the PVDF membranewas dried and then wetted with methanol for a few seconds followed bydeionized water for 1 minute. The PVDF membrane was then soaked for 1 to2 minutes in a protein staining solution consisting of 0.05% (w:v)copper phthalocyanine-3,4′4″4′″-tetrasulfonic acid tetrasodium salt(CPTS) in 0.1% TFA. After staining, the membrane was destained 5 timeswith water (50 mL and 3 min/each wash) and allowed to dry. The stainedthree stained spots were excised from the membrane and transferred to asequencer cartridge for sequencing. The resultant chromatogram andsequencing yields from the first sequencing cycles are shown in FIG. 5and Table 1, respectively.

TABLE 1 Amount Loaded Init. Sequ. (3 ug) Yield Ribonuclease 21 pmol 56%Lysozyme 18 pmol 62% Apomyoglobin 18 pmol 61%

Example 2 Purification and Sequencing of Trypsin Digest of BSA

A tryptic digest of bovine serum albumin (BSA, 10 pmol) containingmethyl violet base B was chromatographed using the system describedabove. Specifically, a 9 μL sample solution was mixed with 1 μL “DyeMark” stock solution (stock=1 mg methyl violet base B per 50 mL aqueous0.1% TFA). The elution gradient was 5%-45% B over 140 min at a flow rateof 4 μL/min. The resultant chromatogram and sequencing data for selectedpeaks are shown in FIG. 6 and Table 2, respectively.

TABLE 2 Locations in BSA Init. Sequ. Peak (Residue Number) Yield 1310-318 21% 2 161-167 24% 3 66-75 40% 4 319-336 N/A* 5 569-580 49% 645-65 50% *Not available.

Example 3 Purification and Sequencing of t-PA Glycopeptides

Glycopeptides from a tryptic digest of t-PA were chromatographed usingthe system described above and then sequenced. Chromatograms of thedigest and the three resolved glycopeptides are shown in FIGS. 7A and7B, respectively. Initial sequencing yields were about 30-40% for 100pmol loaded sample. With reference to FIG. 7B, the three glycopeptidescorresponded to residues 441-449 (peak 1), 163-189 (peak 2), and 102-124(peak 3), as determined by sequence analysis.

Example 4 Purification and Sequencing of Apomyoglobin

A tryptic digest of apomyoglobin (1 pmol) containing methyl violet baseB was chromatographed using the system described above. A chromatogramis shown in FIG. 8. Initial sequencing yields for peak 4 (residues134-139) and peak 6 (residues 64-77) were 42% and 63%, respectively.

Although the invention has been described by way of illustration andexample for purposes of clarity and understanding, it will beappreciated that various modifications can be made without departingfrom the invention. All references and patent applications cited aboveare incorporated herein by reference.

It is claimed:
 1. A blotting apparatus for collecting one or moremolecular components derived from a mixture of components, comprising acapillary liquid chromatography column, an adsorbent collection layer,and means for depositing component-containing eluate from a capillaryliquid chromatography column as a series of discrete, defined-volumemicrodrops, along a region of the adsorbent collection layer.
 2. Theapparatus of claim 1, further including means for monitoring eluate froma capillary liquid chromatography column to detect the presence ofseparated components in the eluate, and a control unit operativelyconnecting the monitoring and depositing means for controlling the flowrate and volume of deposited microdrops.
 3. The apparatus of claim 1,wherein the collection layer is immobile, and said depositing meansinclude a deposition head, for depositing said eluate on the collectionlayer, capable of reciprocating toward and away from a position ofcontact with the collection layer while the deposition head is movedlaterally relative to the collection layer.
 4. The apparatus of claim 1,wherein said depositing means includes (i) a deposition head, fordepositing column eluate on the collection layer, said deposition headbeing capable of reciprocating toward and away from a position ofcontact with the collection layer, and (ii) means for moving saidadsorbent layer laterally relative to the deposition head.